Abstract

BMP2 (bone morphogenetic protein 2) is a multifunctional member of the
transforming growth factor-β family of growth factors. Disruption of BMP2
signaling results in developmental defects, cancers, and other diseases. BMP2
mRNAs are alternatively polyadenylated, resulting in mRNAs with distinct
3′-untranslated regions. The longer mRNA contains additional putative
binding sites for post-transcriptional regulatory factors, including
micro-RNAs. We combined functional assays with computational analyses of
emerging genome data to define site- and species-specific polyadenylation
determinants. In all mouse and human cell lines tested, shorter mRNAs
resulting from using the first polyadenylation signal (PA1) were more abundant
than mRNAs from the second signal (PA2). However, the PA1/PA2 usage ratios
were 2–3-fold higher in human than in mouse cells. Expression of human
BMP2 constructs in mouse cells and mouse constructs in human cells
showed that cis-regulatory elements direct species-specific 3′
processing of BMP2 transcripts. A 72-nucleotide region downstream of PA2 in
the mouse sequence contains two novel cis-acting elements previously
hypothesized to regulate polyadenylation in a bioinformatics analysis.
Mutations that humanized the mouse-specific elements lowered the affinity for
cleavage stimulation factor CstF64 and significantly weakened the PA2 signal
relative to the PA1 signal. Thus, we have experimentally defined for the first
time cis-regulatory elements that control a species-specific
difference in the 3′-end processing of BMP2 and potentially of other
genes